We are pursuing an HIV vaccine approach based on replication-competent Adenovirus (Ad)-recombinants. The rationale is based on the fact that live attenuated vaccines historically have been the most protective, eliciting essentially life-long immunity. Examples include vaccines for small pox, polio, measles, and yellow fever. We conduct pre-clinical vaccine studies in rhesus macaques and challenge with SIV, a system that appropriately models HIV-infection of humans. We use a prime-boost strategy, first immunizing with a replicating adenovirus (Ad) vector carrying an HIV/SIV gene(s) followed by a boosting with HIV/SIV envelope protein. Ad replicates in epithelial cells that line mucosal inductive sites, and elicits strong, persistent cellular immunity at mucosal effector sites as well as in the blood. A study evaluating the biodistribution of the replicating vector showed that regardless of the immunization route (sublingual, intranasal/intratracheal, intravaginal, or intrarectal), genes inserted into the vector were expressed in macrophages in lung and rectal tissue, and subsequently in myeloid dendritic cells of the lung. Expression persisted in rectal tissue up to 25 weeks post-immunization. This targeting of macrophages and professional antigen presenting cells and the persistent expression provide potent immunogenicity, both systemically and mucosally. In line with the similar biodistribution, comparable SIV-specific immunity was elicited by all mucosal immunization routes. We have also shown that the replicating Ad vector elicits a spectrum of cytokine/chemokine responses, which contribute to elicitation of adaptive immunity. Additionally, because non-neutralizing antibody responses have been shown to be contributors to protective efficacy, we have undertaken studies of effector cells which mediate some of these responses. During the period covered by this annual report we showed that following Gag-stimulation of peripheral blood mononuclear cells of SIV-infected rhesus macaques, NK cell responses were induced in animals that controlled SIV infection but not in macaques that did not. The NK cell responses were found to be dependent on antigen-specific IL-2 production by CD4+ central memory T cells. These results suggest that control of disease progression in SIV controlling macaques is associated with co-operation between antigen-specific CD4+ T cells and NK cell effector function and highlight the importance of such cell-to-cell cooperation in adaptive immunity. Current studies are evaluating this co-operative interaction in macaques undergoing treatment. We have also evaluated the effects of immune modulation in SIV infected macaques treated with a novel drug that targets cells expressing high levels of PD-1. High-level T-cell expression of PD-1 during SIV infection is correlated with impaired proliferation and function. We found that continued immune modulation targeting PD-1hi cells during and post-anti-retroviral therapy helped maintain lower viremia and a favorable T-cell/Treg repertoire, while modulating antigen-specific responses.